Methods for the isolation of heparin-binding brain mitogens

ABSTRACT

A group of growth factors, designated heparin-binding brain mitogens (HBBMs), is disclosed. The HBBMs are isolated from brain tissue by a sequence of purification steps. The growth factors may be useful in the promotion of angiogenesis, such as in the promotion of wound healing, bone healing and in the treatment of burns, as well as in promoting the formation, maintenance and repair of tissue, in particular, neural tissue.

This is a divisional of co-pending application Ser. No. 07/787,692,filed on Nov. 1, 1991, U.S. Pat. No. 5,171,842, which is a continuationof application Ser. No. 07/147,835, filed on Jan. 25, 1988, nowabandoned.

SUMMARY OF THE INVENTION

This invention relates to a group of novel protein growth factors whichare believed to promote angiogenesis and, therefore, should be useful inwound healing, bone healing and the treatment of burns. The proteinsinduce mitogenesis in endothelial cells and, as such, may be consideredto be growth factors for those cells. The proteins are also believed topromote the formation, maintenance and repair of tissue, in particular,neural tissue.

The proteins are isolated from brain cells and may each be termed aheparin-binding brain mitogen (HBBM). The proteins are single chain andhighly basic. Three such proteins have been isolated from bovine brain,and have been designated HBBM-1, HBBM-2 and HBBM-3, with molecularweights of 18, 16 and 15 kd, respectively. The proteins possess a common19 amino acid N-terminal sequence which differs from that of other knownproteins.

The same three HBBMs have also been isolated from human brain tissue andhave the same N-terminal sequence and the same type of mitogenicactivity as bovine HBBMs. Rat and chicken brains have also been found tocontain HBBMs.

The proteins are isolated and purified from brain tissue by acombination of steps, which include extraction from the tissue, heparinaffinity chromatography, and cation-exchange chromatography. Hydrophobicinteraction chromatography may be used as an auxiliary method ofpurification.

BACKGROUND OF THE INVENTION

Numerous protein growth factors have been isolated and characterized inrecent years. These growth factors include epidermal growth factor,fibroblast growth factors, insulin-like growth factors, transforminggrowth factors, platelet-derived growth factor and interleukins. Forexample, fibroblast growth factor ("FGF") was first purified byGospodarowicz in 1975 from bovine pituitary and had an estimatedmolecular weight of 13,300 daltons (Reference 1).

FGF was later purified from bovine brain (2). FGF can be isolated ineither an acidic ("aFGF") or basic ("bFGF") form, depending on theisolation procedures used (3,4). A complete amino acid sequence forbovine pituitary bFGF (5) and bovine and human brain aFGF (6,7) has beenpublished, together with the N-terminal sequences for bovine and humanbrain bFGF (5). The N-terminal sequences for bovine pituitary and bovinebrain bFGF are identical (5,8).

It has now been found that, when the FGFs are purified from brain tissueusing heparin-Sepharose affinity chromatography (9,10), a significantquantity of unknown proteins may also be present. Because proteins thatbind with particularly high affinity to heparin are rare, further studyof these unknown proteins was undertaken. This investigation revealedthese proteins to be the HBBMs, which differ from the FGFs in theirN-terminal sequence and amino acid compositions.

Accordingly, it is an object of this invention to isolate, purify andcharacterize the HBBMs from brain tissue. It is a further object of thisinvention to establish the physiological activity of the HBBMs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the fractionation of 400 ml of the 0.6M NaCl eluate fromcation exchange chromatography using heparin-Sepharose affinitychromatography. The horizontal bar A indicates the fractions containingthe HBBMs which were thereafter rechromatographed for furtherpurifaction. The horizontal bar B indicates the fractions containingaFGF, as determined by reverse-phase HPLC analysis.

FIG. 2 depicts the results of Mono-S cation exchange chromatography on20 ml of the material eluted from the heparin-Sepharose affinitychromatography (as indicated by the horizontal bar A, FIG. 1). Arrowsindicate the elution positions of aFGF and bFGF standards. The numbersin the chromatogram refer to HBBM-1, -2, and -3. The horizontal barindicates the fractions containing the HBBMs which were used as a poolor individually for further purification/characterization.

FIG. 3 depicts the reverse-phase HPLC of HBBMs. An aliquot of the poolof Mono-S-purified HBBMs (peaks 1-3, as indicated by the horizontal barin FIG. 2) was subjected to reverse-phase HPLC. Individual peaks wereidentified and assigned to specific Mono-S fractions by analyzingaliquots of Mono-S chromatography fractions (as indicated by sections ofthe horizontal bar in FIG. 2) in independent reverse-phase HPLC runs.Peaks 1-3 refer to HBBM-1, HBBM-2, and HBBM-3, respectively.

FIG. 4 depicts the analysis on SDS-PAGE of the HBBMs as follows: Lane 1:Protein standards (lysozyme, 14.4 kD; trypsin inhibitor, 21.5 kD;carbonic anhydrase, 31 kD; ovalbumin, 45 kD, serum albumin, 66.2 kD);Lane 2: HBBM-1; Lane 3: HBBM-2; Lane 4: HBBM-3.

FIG. 5 depicts the rechromatography of HBBM-3 on a Mono-Scation-exchange column. The fractions containing HBBM-3 from thechromatography depicted in FIG. 2 were pooled, diluted three times withstarting buffer, and the diluted samples chromatographed on a Mono-Scolumn, under conditions identical to those shown in FIG. 2. Aliquots offractions were tested for their ability to stimulate the growth ofbovine aortic arch endothelial cells (histogram). Retention times ofaFGF and bFGF are shown with arrows. A horizontal bar marks thefractions which thereafter were used for concentrating HBBM-3.

FIG. 6 depicts the biological activity of HBBM-3 and HBBM-1 by means ofdose response analysis and comparison with FGF activity. The ability ofHBBM-3 and HBBM-1 to stimulate the growth of bovine aortic archendothelial cells was tested. Upper panel: HBBM-3; Lower panel: HBBM-1.In each panel, solid line: HBBM; dashed line: aFGF; dotted line: bFGF.

FIG. 7 depicts the chromatography of HBBM-3 on an hydrophobicinteraction column. An aliquot of Mono-S-concentrated HBBM-3 wassubjected to HIC chromatography. Arrows indicate the retention times ofaFGF and bFGF under identical chromatographic conditions. Aliquots ofcolumn fractions were tested for their ability to stimulate bovineaortic arch endothelial cell proliferation. The results are indicated bythe number of cells grown in each test well.

DETAILED DESCRIPTION OF THE INVENTION

The novel growth factors of this invention are single chain, basic,heparin-binding brain mitogens. HBBMs have been identified in the braintissues of all species tested, which include human, bovine, rat andchicken. Based on this distribution, it is expected that other specieswill also contain HBBMs.

The N-terminal sequences of the HBBMs differ markedly from thosepublished for bovine or human brain aFGF and bFGF (5,6,7). TheN-terminal sequences of the first 19 amino acids have been found to beidentical for human and bovine HBBMs. The N-terminal sequences are asfollows:Gly-Lys-Lys-Glu-Lys-Pro-Glu-Lys-Lys-Val-Lys-Lys-Ser-Asp-Cys-Gly-Glu-Trp-Gln.The rat sequence is identical with the exception of the cysteine residuein position 15, because a determination of the presence or absence ofcysteine was not conducted. However, the fact that no other amino acidwas identified at position 15 is consistent with the presumed presenceof cysteine. To date, the first ten N-terminal residues of chicken HBBMshave been sequenced; they are identical to those of human, bovine andrat HBBMs.

The HBBMs have been found in three forms in tissue from bovine brain.HBBM-1, HBBM-2 and HBBM-3 have molecular weights of 18, 16 and 15 kD,respectively. Their amino acid compositions (determined as hereinafterdescribed) are set forth in Table I below and also differ from the aminoacid compositions of the bovine brain FGFs (5,6,7).

Based on the molecular weights and the identical N-terminal sequences,it is concluded that the three HBBMs probably differ at their C-termini.The available data strongly suggest that HBBM-2 and HBBM-3 areC-terminally truncated forms of HBBM-1, lacking approximately 13 and 29amino acids, respectively.

It was found that the ratios of the three forms varied between differentisolation batches. However, on average, based on quantitative amino acidanalysis, the overall isolation yield of HBBMs was estimated atapproximately 30, 10 and 40 μg/kg brain tissue for HBBM-1, HBBM-2, andHBBM-3, respectively. The ratios may depend on variables during tissuestorage and extraction, in particular proteolysis. Proteolysis duringtissue extraction may cause carboxy-terminal truncation of HBBM-1, thelargest of the HBBM forms, which would yield HBBM-2 and HBBM-3 invarying amounts.

The process for isolating the novel HBBMs of this invention insubstantially pure form from a source of brain tissue comprises thesequence of steps of:

(a) extraction from the source tissue;

(b) cation-exchange chromatography;

(c) heparin affinity chromatography;

(d) cation-exchange chromatography; and, optionally,

(e) hydrophobic-interaction chromatography.

The extraction is accomplished by treating the tissue sequentially with0.15M ammonium sulfate, adjusting to pH 4.5 with hydrochloric acid,stirring and centrifugation, treating with ammonium sulfate after the pHis adjusted to 6-6.5 with sodium hydroxide, stirring and centrifugation,followed by dialysis and centrifugation.

The first cation-exchange chromatography step comprises batch adsorptionon a carboxymethyl-Sephadex column, followed by washing with 100 mMsodium phosphate buffer at pH 6 and elution with 100 mM sodiumphosphate, pH 6.0/0.6M NaCl.

The heparin affinity chromatography is performed on a heparin-Sepharose(Pharmacia) column by washing with a buffer containing 10 mM Tris-HCl,pH 7.0/0 6M NaCl, followed by elution with a linear gradient of from 0.6to 2.0M NaCl in 10 mM Tris-HCl, pH 7.0.

The second cation-exchange chromatography step is performed on a Mono-S(Pharmacia) column equilibrated and, after loading of the sample, washedwith 50 mM sodium phosphate, pH 6.8, followed by elution of HBBMs with alinear gradient of from 0 to 0.6M NaCl in 50mM sodium phosphate, pH 6.8.

The hydrophobic-interaction chromatography step comprisespreequilibrating a HIC column (LKB Ultropac-TSK-Phenyl-5PW) with abuffer of 100 mM sodium phosphate, pH 7.0, and 1.5M sodium sulfate,followed by elution with a linear gradient of from 1.5 to 0.6M sodiumsulfate.

Although the HBBMs may be separated easily and quantitatively from theFGFs by reverse-phase HPLC, this procedure reduces the biologicalactivity of the FGFs due to the conditions used in reverse-phase HPLC.Therefore, in order to separate and isolate the HBBMs and the FGFs inbiologically active form for comparative testing, the process set forthabove was developed. The use of reverse-phase HPLC was limited to thetesting of small aliquots of fractions from heparin-Sepharose affinitychromatography and cation-exchange chromatography for the presence ofHBBMs and FGFs. The reverse-phase HPLC steps were performed on a C4column (The Separations Group) eluted with a shallow gradient ofacetonitrile in 0.1% trifluoroacetic acid.

The biological activity of the HBBMs has been established by testsevidencing the induction of mitogenesis in endothelial cells. AlthoughHBBM-2 was not tested, the activity demonstrated for HBBM-1 and HBBM-3suggests that HBBM-2 will also possess this activity. The testing wasperformed on bovine aortic arch endothelial cells.

Three separate tests were performed, both to confirm the existence ofmitogenic activity and to demonstrate that the activity was due to theHBBMs and not to the FGFs.

First, the eluent fractions from Mono-S chromatography which containedHBBM-3 were pooled, rechromatographed on a Mono-S column and then testedfor their ability to stimulate the growth of bovine vascular endothelialcells. For comparison, samples of aFGF and bFGF were also tested. FIG. 5presents the results of this test.

Next, the available HBBM-3 fractions were rechromatographed for sampleconcentration on a Mono-S column using a steeper gradient and lower flowrate. The HBBM-3 was then compared with aFGF and bFGF in dose-responseanalyses. The upper panel of FIG. 6 presents the results of this test.This procedure was then repeated for HBBM-1. Those results are presentedin the lower panel of FIG. 6.

Finally, HBBM-3 fractions concentrated by Mono-S chromatography wereplaced on an hydrophobic interaction column, which has differentselectivities than a Mono-S column. FIG. 7 presents the results of thistest.

These tests established that the HBBMs stimulate the proliferation ofcultured bovine vascular endothelial cells in a dose-dependent manner.The ED₅₀ was approximately 20 ng/ml for HBBM-3 and 180 ng/ml for HBBM-1.The ED₅₀ was calculated as follows: ED₅₀ =conc [cell no.(dose=0)+cellno.(dose=max)/2]. These mitogens were comparable to the FGFs in terms ofbiological activity and high affinity to heparin. HBBM-1 and HBBM-3 areeach equipotent to aFGF. The ED₅₀ are in the range of 50-150 ng/ml.

The test results also make plain that the biological activity associatedwith HBBM-3 was genuine and was not the result of contamination with anFGF. HBBM-3 and the FGFs are clearly separated by highly resolutiveMono-S chromatography, as shown in FIG. 5, and are also clearlyseparated by a second high resolution technique with differentselectivities, hydrophobic interaction chromatography, as shown in FIG.7.

Moreover, the HBBMs did not contain measurable quantities of aFGF asdetermined by quantitative reverse-phase HPLC. As further evidence thatthe activity was due to HBBMs, the HBBMs were not cross-reactive in animmuno-dot assay using polyclonal antibodies raised against syntheticpeptides corresponding to the N-terminal 15 residues of aFGF (1-15) andbFGF (30-50), the latter yielding an antibody which cross-reacts withaFGF (antibodies provided by A. Baird, Salk Institute; growth factornomenclature according to refs. 5,7). Finally, the concentrations ofHBBM and aFGF preparations were carefully determined by quantitativeamino acid analysis as a basis for potency comparisons (FIG. 6). Basedon those measurements, an inherently inactive HBBM-3 would have to becontaminated with an equal amount of aFGF in order to produce thebiological response seen. The results rule out such a contamination. Insummary, the results of those studies clearly show that the activity ofHBBM-3 is not a consequence of aFGF contamination.

Several lines of evidence indicate, furthermore, that HBBM-3 activity isnot due to bFGF contamination. In theory, a few percent contamination ofHBBM-3 with bFGF would suffice to produce the activities observed.However, such a contamination is not indicated, because HBBMs and bFGFare widely separated on heparinSepharose chromatography. Moreover,HBBM-3 and bFGF are also well-separated in Mono-S chromatography (FIG.5). Finally, when HBBM-3 was subjected to chromatography on a thirdhighly resolutive system, hydrophobic interaction chromatography, it wasevident that biological activity was still associated with HBBM-3 andnot with the well-separated bFGFs (FIG. 7). There is no evidence thatthe two entities would copurify, even in trace amounts, in three widelydisparate and resolutive chromatographic systems.

Based on those results, it is concluded that the activity of HBBM-3 isgenuine and not the result of contamination with the knownheparin-binding FGFs.

The HBBMs are further distinguished from the FGFs by a lack of aminoacid sequence homology between the two groups of proteins. In additionto the difference in N-terminal sequences described earlier, thepresently available sequence information (approximately 75 of 130residues of HBBM-3) shows no sequence homology to the published 146residue sequence of the FGFs (5).

The mitogenic activity of human HBBMs was generally found to beindistinguishable from that of bovine HBBM, although the human mitogenswere less extensively characterized owing to limited quantities ofmaterial available. This is consistent with other findings: identicalamino-terminal sequence, presence of three forms, and retention behavioron ion-exchange, heparin-Sepharose, and reverse-phase chromatography.

The identity of N-terminal sequences among the various sources of theHBBMs suggests that evolutionary pressure may have prevented mutations,at least during the evolution from birds to mammals. Sequenceconservation as a result of evolutionary pressure is strongly suspectedwith many well-known, biologically active proteins that are highlyhomologous between species.

The HBBMs were not present in bovine kidney tissue that was extractedusing the procedure for brain tissue Therefore, the HBBMs are novelproteins which may also play a role in the formation, maintenance and/orrepair of tissue, in particular, neural tissue.

The bases for this statement about the role of HBBMs are the following:(1) the HBBMs have the same biological and heparin-binding activities asaFGF; (2) the HBBMs are brain-specific; (3) higher amounts of the HBBMsthan aFGF are found in the brain; and (4) aFGF and bFGF are known tohave very prominent neural activities, such as neurotrophic (neuronsurvival) activity in vivo and in vitro, they are mitogenic forneuroblasts and glial cells, they promote neurite outgrowth and inducebrain-specific protein synthesis.

Although the protein structures of the HBBMs and the FGFs aredissimilar, their similarity in terms of heparin binding and biologicalactivity suggests that both groups of mitogens act through a similarmechanism. For example, the mitogens could bind to cell surface orextracellular, matrix-associated heparinlike structures.

Therapeutic compositions in accordance with this invention includeHBBMs, either singly or in mixtures, dispersed in a conventionalpharmaceutically acceptable liquid or solid carrier. The therapeuticcompositiohs may be administered topically in the form of creams,lotions and so forth, or orally in such forms as tablets, capsules,dispersible powders, granules or suspensions, or parenterally in theform of sterile injectable solutions or suspensions. These therapeuticcompositions may be administered to human or veterinary patients topromote angiogenesis and the repair and maintenance of neural tissue.

EXAMPLE

In this example, the isolation and characterization of bovine HBBM areset forth in detail. Results using human material were very similar andwill not be described in detail unless differences from bovine materialwere found.

1) Tissue Extraction

Human brains were obtained less than 24 hours post-mortem from theDepartment of Pathology, University of Zurich. Bovine brains wereobtained at a slaughterhouse. Tissues were frozen immediately afterreceipt, stored at -80° C., and processed within two weeks afterreceipt. Batches of 3-5 kg of brain tissue were extracted at a time.

Brain tissue was extracted following the procedure developed byGospodarowicz and co-workers (2) as described (6,10): Frozen brains werecrushed with a hammer. Six-hundred gram portions of tissue werehomogenized in 1.2 1 of 0.15M ammonium sulfate for 3 minutes in a WaringBlender. The pH of the homogenate was adjusted immediately to pH 4.5with concentrated HCl and the mixture was further homogenized using aPolyton homogenizer until the tissue was finely dispersed. Thehomogenate was then extracted by stirring of the suspension for 2 hoursat 4° C. followed by centrifugation at 4° C. for 60 minutes at either11,500 rpm (GSA rotor) or 9,000 rpm (GS-3 rotor) to remove cells anddebris. The supernatant was adjusted to pH 6-6.5 with NaOH, ammoniumsulfate (230 g/l) was added slowly, and the resulting suspension wasstirred for at least 30 minutes at 4° C. and centrifuged again asdescribed above. The pellet was discarded. More ammonium sulfate (300g/l) was slowly added to the supernatant, the suspension was stirred forat least 30 minutes at 4° C. and centrifuged as above. The resultingpellet was dissolved in cold water (100 ml per kg of starting material)and dialysed at 4° C. for 20 hours against 20 1 of water using aSpectrapor membrane (molecular weight cut-off 6-8 kD, diameter 31.8 mm).The dialysate was centrifuged again to remove precipitated material andthe supernatant subjected to chromatographic purification (see below)after determining its conductivity.

2) Cation Exchange Chromatography

The tissue extract resulting from the above procedure (approximately 150ml for each kg of brain tissue) was subjected to batchadsorption/elution by cation exchange chromatography as follows: Thesample was diluted with water as required to bring the conductivitybelow that of a 0.1M sodium phosphate buffer (pH 6)/0.15M NaCl solution.The sample was then loaded onto a column of carboxymethyl-Sephadex(5.5×3 cm) which was preequilibrated with 100 mM sodium phosphate (pH6)/0.15M Cl. The column was washed with the equilibration buffer and aprotein fraction was eluted with 100 mM sodium phosphate, pH 6.0/0.6MNaCl and collected. All operations were carried out at room temperatureand a flow rate of 500 ml/hour.

3) Heparin-Sepharose Affinity Chromatography

The 0.6M NaCl eluate from cation exchange chromatography was loaded on aheparin-Sepharose column (Pharmacia, 5×1.5 cm) at a flow rate of 125ml/hour. The column was washed with 200 ml of a buffer containing 10 mMTris-HCI, pH 7.0/0.6M NaCl until the absorbance of the column eluate at280 nm became negligible. Protein bound to the column was eluted with a120-minute linear gradient from 0.6M to 2M NaCl in 10 mM Tris-HCl, pH7.0 at a flow rate of 35 ml/h. Chromatography was performed at roomtemperature using a LKB peristaltic pump and a low pressure LKBprogrammable gradient former. Fractions of 1.4 ml were collected andaliquots subjected to bioassay.

The results of this Heparin-Sepharose chromatography are shown inFIG. 1. The fractions subjected to further purification, using stepsdescribed below, are indicated by the horizontal bar A. The fractionscontaining aFGF eluted between 1.1-1.3M NaCl (as determined by HPLCanalysis of individual heparin-Sepharose column fractions; data notshown) and correspond to the shoulder of the larger peak eluting at1-1.2M NaCl, and to the horizontal bar B. The reverse-phase HPLCanalysis was conducted on a C4 column (25×0.46 cm, 5 um particle size,300 angstroms pore size, The Separations Group, Hesperia, Calif.). Theproteins were eluted in a shallow gradient of acetonitrile (10%/hour) in0.1% trifluoroacetic acid at a flow rate of 0.7 ml/minute. Theheparin-Sepharose chromatography was performed at room temperature.

4) Mono-S Cation Exchange Chromatography

In order to separate the contaminating aFGF from the other proteinmaterial, fractions eluting at 1-1.2M NaCl were pooled, dilutedsufficiently with a buffer containing 50 mM sodium phosphate, pH 6.8 (toreduce their ionic strength to approximately that of the diluent) andsubjected to cation-exchange chromatography on a Mono-S column(Pharmacia) equilibrated with 50 mM sodium phosphate, pH 6.8. Thismaterial was pumped onto a Mono-S column (Pharmacia) equilibrated with50 mM sodium phosphate, pH 6.8. After washing the column with the samebuffer until the absorbance at 210 nm reached a minimum value, proteinwas eluted with a gradient from 0 to 0.6M NaCI in 50 mM sodiumphosphate, pH 6.8.

Several well-discernible peaks were eluted from the Mono-S column underthese conditions, as shown in FIG. 2. Each peak was analyzed byreverse-phase HPLC using LKB HPLC equipment at room temperature with aflow rate of 0.7 ml/minute.

The first and quantitatively minor peak which eluted from the Mono-Scolumn at 0.3M NaCl corresponded to aFGF as evidenced by co-elution witha reference standard of authentic aFGF in the same system underidentical conditions (see arrow in FIG. 2), as well as by co-elution inreverse-phase HPLC on a previously-described C4 column, eluted underhighly resolutive, shallow gradient conditions (data not shown). Thepresence of aFGF in this Mono-S column fraction was expected, becausethe originating fraction from heparin-Sepharose chromatography is knownto contain aFGF. The major part of the material eluted from the Mono-Scolumn at approximately 0.45-0.65M NaCl as a relatively well-resolvedtriplet of peaks. Since those peaks were determined to contain HBBM, ashereinafter described, they were designated as HBBM-3, HBBM-2, andHBBM-1, in the order of their elution (FIG. 2). The elution behavior ofhuman HBBM on a Mono-S column is similar in general, but has been lesswell studied than bovine HBBM.

The HBBMs are clearly distinguishable by chromatographic retention notonly from aFGF but also bFGF (FIG. 2).

Moreover, when analyzed by reverse-phase HPLC (FIG. 3), the HBBMs werealso found to differ clearly from the FGFs with respect to retentiontimes (data not shown). Finally, reverse-phase HPLC separated all threeHBBMs from each other (FIG. 3) and thus provided a means for theirpreparation in high purity (as needed for structural characterization)and for relatively unambiguous identification.

Human brain also yielded three forms of HBBMs with reverse-phase HPLCelution patterns identical to those of their bovine counterparts.

5) Hydrophobic Interaction Chromatography

Fractions from Mono-S chromatography containing samples of interest weremade 1.5M in sodium sulfate and applied to an HIC column (LKB UltropacTSK-Phenyl-5PW, 7.5×75 mm) which was preequilibrated with a buffer of100 mM sodium phosphate, pH 7.0/1.5M sodium sulfate. Protein waschromatographed at room temperature using a 30-minute linear gradientfrom 1.5 to 0.6M sodium sulfate at a flow rate of 1.0 ml/min.Hydrophobic interaction chromatography is a separation system withselectivities quite different from Mono-S chromatography. Consequently,the separation of the HBBMs from the FGFs by HIC (as shown in FIG. 7),reinforces the result of Mono-S chromatography (as shown in FIG. 5) thatthe HBBMs are different chemical entities from the FGFs.

6) Amino Acid N-Terminal Sequence Analysis

Usually, sequence analyses were conducted without chemical modificationof the proteins. However, as a preliminary step in conducting someanalyses of the amino acid N-terminal sequence of the HBBMs, thecysteine residues of the HPLC-purified proteins were treated accordingto the procedure of Gautschi-Sova et al. (6). Briefly, the cysteineresidues were reduced with a five-fold molar excess of dithiothreitoland alkylated by carboxymethylation using a three-fold molar excess ofiodo-[2-¹⁴ C]-acetic acid.

The amino acid N-terminal sequence analyses of proteins (100-500 pmol)were performed on an Applied Biosystems (Foster City, Calif.) Model 470Agas/liquid phase protein microsequenator as described by Esch et al.(5). In addition, phenylthiohydantoin (PTH) derivatives of amino acidswere identified by reverse-phase HPLC on an Applied Biosystems Model120A On-line PTH amino acid analyzer. Both procedures were carried outaccording to protocols from the instrument manufacturer using chemicalssupplied by Applied Biosystems.

The N-terminal sequences of all three HBBMs were found to be identicalto each other for the first 19 amino acids. The sequences weredetermined asGly-Lys-Lys-Glu-Lys-Pro-Glu-Lys-Lys-Val-Lys-Lys-Ser-Asp-Cys-Gly-Glu-Trp-Gln.Human HBBMs (probably analyzed as a mixture) were found to possess thesame N-terminal sequence as bovine HBBMs.

While sequencing data clearly indicate that the three bovine HBBMproteins are structurally related to each other, amino acid compositionsand molecular weights further suggest that those proteins may differ inthe carboxy-terminal region. All data are compatible with thealternative interpretations that either HBBM-2 and HBBM-3 arecarboxy-terminally truncated forms of HBBM-1, lacking approximately 13and 29 amino acids, respectively, at their carboxy-terminals.

7) Molecular Weights of HBBMs

Protein samples were analyzed using sodium dodecyl sulfatepolyacrylamide gel electrophoresis ("SDS-PAGE") as described byGospodarowicz et al. (9). Briefly, molecular weight determinations wereperformed by SDS-PAGE as follows: Aliquots containing 40-80 ng ofprotein were added to the sample buffer composed of 30% (v/v) glycerol,0.2M dithiothreitol, 4% (w/v) sodium dodecyl sulfate, 4 mM EDTA, and 75mM Tris-HCl, pH 6.8. Samples were boiled for 3 minutes and then appliedto a 20% polyacrylamide gel slab (1.5 mm) with a 3% stacking gel.Electrophoresis under non-reducing conditions was performed in identicalfashion except that dithiothreitol was omitted from the sample buffer. Aprotein standard mixture containing lysozyme (14.4kD), trypsin inhibitor(21.5kD), carbonic anhydrase (31 kD), ovalbumin (45 kD) and serumalbumin (66.2 kD) was also applied to each gel. SDSPAGE revealed themolecular weights of HBBM 1, -2, and -3 to be 18, 16 and 15 kD,respectively (FIG. 4). The molecular weights did not differsignificantly regardless of whether the samples were electrophoresed inthe presence or absence of reducing agent, indicating that the proteinsare single chain polymers. The molecular weights of the human HBBMs havenot yet been determined by SDS-PAGE.

8) Amino Acid Composition Analysis

A 10-20 pmol protein sample was hydrolyzed according to the highsensitivity methodology of Bohlen and Schroeder (11). Briefly, theprotein sample was added to a hydrolysis tube and dried in vacuo. Fiftyμl constant boiling HCl containing 2% (v/v) thioglycollic acid wereadded. The tube was then evacuated with high vacuum (less than 50mmTorr) after freezing the sample in liquid nitrogen. The sample was thenallowed to melt while under vacuum and the tube was flame-sealed.

The tube was hydrolyzed by heating at 110° C. for 20 hours. Afterhydrolysis, the tube was opened, dried in vacuo, and the residuedissolved in 130 μl citrate buffer (0.067 sodium citrate, pH 2.20) priorto loading on the cation exchange chromatography column.

The protein hydrolysates were chromatographed on a Chromakon 500 aminoacid analyzer (Kontron, Zurich, Switzerland) equipped with apolystyrene-based cation exchange column and an o-phthalaldehydefluorescence detection system for high-sensitivity detection (11).

Quantitation of amino acids was by the external standard method using anamino acid standard mixture. Based on quantitative amino acid analysis,the overall isolation yield of HBBMs was estimated at approximately 30,10, and 40 μg/kg brain tissue for HBBM-1, HBBM-2, and HBBM-3,respectively.

Amino acid compositions of the three HPLC-purified proteins are shown inTable I.

                  TABLE I                                                         ______________________________________                                        Amino acid compositions of heparin-binding brain mitogens                                     HBBM-1 HBBM-2   HBBM-3                                        ______________________________________                                        Molecular weight.sup.1                                                                          18,000   16,000   15,000                                    Order of elution                                                              reverse-phase HPLC                                                                              1        2        3                                         Mono-S            3        2        1                                         Amino acid (number of residues)                                               Asparagine & aspartic acid                                                                      10       10       9                                         Threonine         14       14       13                                        Serine            9        9        6                                         Glutamine & glutamic acid                                                                       23       20       17                                        Proline           nd.sup.2 nd       nd                                        Glycine           16       15       13                                        Alanine           10       8        8                                         Cysteine          nd       nd       nd                                        Valine            4        4        4                                         Methionine        1        1        1                                         Isoleucine        2        2        2                                         Leucine           8        .sup. 7.sup.3                                                                          8                                         Tyrosine          2        2        1                                         Phenylalanine     2        2        2                                         Histidine         1        1        1                                         Lysine            35       28       23                                        Tryptophan        3        nd.sup.3 3                                         Arginine          8        .sup. 7.sup.3                                                                          8                                         ______________________________________                                         .sup.1 as determined by SDSPAGE                                               .sup.2 nd: not determined                                                     .sup.3 in accordance with the hypothesis of Cterminal truncation, it is       believed that the following are the proper values for HBBM2: Leucine 8,       Tryptophan 3, Arginine 8.                                                

Amino acid compositions are calculated from 3-5 determinations.

Amino acid compositional data agree with the elution order of the threeproteins on Mono-S: the least basic protein (HBBM-3) elutes first, whilethe most basic protein (HBBM-1) elutes last. Expected molecular weightscalculated from amino acid analyses are somewhat lower than thosedetermined by SDS-PAGE. The discrepancies may be accounted for byproline and cysteine residues which were not quantitated by amino acidanalysis. However, based on the N-terminal sequence analysis, it isknown that proline and cysteine are in fact present. Amino acid analysesof human HBBMs are not yet available.

9) Biological Activity

The ability of certain of the HBBMs to induce mitogenesis in endothelialcells was tested by measuring the effect of those proteins on theproliferation in vitro of bovine aortic arch endothelial cells which hadbeen cultured as described by Bohlen et al. (2,10). Briefly, the bovineendothelial cells were seeded with column fractions obtained asdescribed below at low density (10,000-20,000 cells/35 mm dish) inDulbecco's modified Eagle's medium containing 10% calf serum (Hyclone,Sterile Systems, Logan, Utah). Cultures were grown for 5 days in thepresence of various concentrations of column fraction aliquots (added ondays 0 and 2) and then counted in a Coulter particle counter. Thebiological activities of the proteins tested are indicated in the numberof vascular endothelial cells grown in each test well for each fractionin FIGS. 5-7.

In particular, activity of the HBBMs was compared with that of the FGFs.Using published procedures (9,12), aFGF and bFGF were isolated and theirauthenticity was verified by N-terminal sequence analysis and molecularweight determination (SDS-PAGE).

The eluant fractions corresponding to the peak for HBBM-3 from Mono-Schromatography, as shown in FIG. 2, were pooled, diluted three timeswith starting buffer and rechromatographed on the same system. Aliquotsof these fractions were tested as described above for their ability tostimulate the growth of bovine vascular endothelial cells. Comparativetests were run with aFGF and bFGF. The results, as shown in FIG. 5,indicated that HBBM-3 stimulated mitogenic activity for bovineendothelial cells. Furthermore, the HBBM-3 and its activity wereseparable from the FGFs.

In further test of activity, the available HBBM-3 fractions wereconcentrated on a Mono-S column using the same buffer system asdescribed for FIG. 2, but with a steeper gradient (0-1.0M NaCl in 20minutes) and lower flow rate (0.4 ml/minute). Comparative dose-responseanalyses were run with aFGF and bFGF.

The upper panel of FIG. 6 indicates that HBBM-3 stimulated bovine aorticendothelial cells in a dose-dependent manner. The ED₅₀ was in the orderof 20-50 ng/ml, with the minimally stimulating dose being approximately3 ng/ml. The dose-response curves of HBBM-3 and aFGF wereindistinguishable, both qualitatively and quantitatively, under theassay conditions used. The response of HBBM-3 appeared to bedistinguishable from that of bFGF. In the assay system used, bFGFpossessed much higher potency and apparently higher intrinsic activitythan HBBM-3. It should be cautioned, however, that the question ofintrinsic activity of HBBM-3 could not be addressed adequately in thistest because doses sufficiently high to allow assessment of theintrinsic activity of HBBM-3 could not be used in the assay due tolimitations with respect to the amount of salt that could be added tothe cells without adverse effects on cell growth. Preliminary resultsindicate, however, that the intrinsic activity of HBBM-3 is identical tothat of aFGF under the assay conditions used.

The dose-response analysis was repeated for HBBM-1. The results, asshown in the lower panel of FIG. 6, indicate that HBBM-1 was alsobiologically active in the same test system. Human HBBM, comprising amixture of HBBM-1, -2 and -3, was tested in the same manner and wassimilarly active (data not shown). However, results have not yet beenobtained for individual fractions of the human HBBMs. Furthermore,bovine HBBMs were also tested on human umbilical cord endothelial cellsand found to be active (data not shown).

Finally, aliquots of HBBM-3 concentrated by Mono-S chromatography weremade 1.5M in sodium sulfate and placed on an hydrophobic interactioncolumn. The results, as shown in FIG. 7, indicated that HBBM-3 wasactive and was well-separated from both aFGF and bFGF.

REFERENCES

1. Gospodarowicz, D., J. Biol. Chem., 250, 2515-2520 (1975).

2. Gospodarowicz, D., Bialecki, H., and Greenburg, G., J. Biol. Chem.,253, 3736-3743 (1978).

3. Bohlen, P., Baird, A., Esch, F., Ling, N., and Gospodarowicz, D.,Proc. Nat. Acad. Sci., 81 5364-5368 (1984).

4. Thomas, K., Rios-Candelore, M., and Fitzpatrick, S., Proc. Nat. Acad.Sci., 81, 357-361 (1984).

5. Esch, F., Baird, A., Ling, N., Ueno, N., Hill, F., Denoroy, L.,Klepper, R., Gospodarowicz, D., Bohlen, P., and Guillemin, R., Proc.Nat. Acad. Sci., 82, 6507-6511 (1985).

6. Gautschi-Sova, P., Mueller, T., and Bohlen, P. BBRC, 140, 874-880(1986).

7. Gimenez-Gallego, G., Rodkey, J., Bennett, C., Rios-Candelore, M.,DiSalvo, J., and Thomas, K., Science, 230, 1385-1388 (1985).

8. Bohlen, P., Esch, F., Baird, A., Jones, K., and Gospodarowicz, D.,FEBS Lett., 185, 177-181 (1985).

9. Gospodarowicz, D., Cheng, J., Lui, G., Baird, A., and Bohlen, P.,Proc. Nat. Acad. Sci., 81, 6963-6967 (1984).

10. Bohlen, P., Esch, F., Baird, A., and Gospodarowicz, D., EMBO J., 4,1951-1956 (1985).

11. Bohlen, P., and Schroeder, R., Anal. Biochem., 126, 144-152 (1982).

12. Gautschi-Sova, P., Jiang, Z. P., Frater-Schroeder, M., and Bohlen,P., Biochemistry, 26, 5844-5847 (1987).

We claim:
 1. A method for the isolation of an essentially purified andisolated heparin-binding brain mitogen having the N-terminal amino acidsequence H-Gly-Lys-Lys-Glu-Lys-Pro-Glu-Lys-Lys-Val-, wherein saidmitogen is selected from the group consisting of HBBM-1, HBBM-2 andHBBM-3, wherein HBBM-1 has a molecular weight of about 18,000 daltons,HBBM-2 has a molecular weight of about 16,000 daltons and HBBM-3 has amolecular weight of about 15,000 daltons, where said molecular weightsare determined by sodium dodecyl sulfate polyacrylamide gelelectrophoresis under either reducing or non-reducing conditions, whichcomprises the sequence of steps of:(a) extraction from the source tissueto obtain an extract containing the heparin-binding brain mitogen; (b)subjecting the extract from step (a) to a first cation-exchangechromatography, such that the eluate contains the heparin-binding brainmitogen; (c) subjecting the eluate from step (b) to heparin affinitychromatography, wherein the eluate is added to a heparin affinitycolumn, such that the heparin-binding brain mitogen binds to the columnand is then eluted from the column; and (d) subjecting the eluate fromstep (c) to a second cation-exchange chromatography to separate anycontaminating acidic fibroblast growth factor from the heparin-bindingbrain mitogen.
 2. The method of claim 1 which further comprises the stepof hydrophobic-interaction chromatography after step (d).
 3. The methodof claim 1 wherein the eluted fractions of steps (c) and (d) areanalyzed for the presence of heparin-binding brain mitogen byreverse-phase high performance liquid chromatography.
 4. The method ofclaim 3 wherein the source tissue is bovine brain.